Sequence analysis of genes encoding structural and nonstructural proteins of a human group B rotavirus detected in Calcutta, India.

Nucleotide sequences of RNA segments encoding structural proteins(VP4, VP6, and VP7) and nonstructural proteins(NSP1 and NSP3) of a human group B rotavirus CAL-1, which was detected in Calcutta, India, were determined and their relatedness with cognate genes of other group B rotaviruses was analyzed. The CAL-1 genes showed generally high sequence identities (more than 90%) to those of human group B rotavirus, adult diarrheal rotavirus (ADRV) in China, while identities with bovine, murine, and ovine viruses were considerably lower (58-73%). Among RNA segments analyzed, sequence identity of the VP6 gene was relatively high compared with other gene segments. In the CAL-1 VP7 sequence, many characteristics were shared by ADRV, but not by other animal group B rotaviruses. In contrast, VP4 and NSP3 of CAL-1 were single amino acid and 23 amino acids longer than those of ADRV strain, respectively, due to differences of a few nucleotides. These findings suggested that human group B rotaviruses CAL-1 and ADRV might have originated from a common ancestral virus distinct from animal group B rotaviruses reported so far, while some notable sequence differences indicated the distinct nature of these viruses.

Distribution of aminoglycoside resistance genes in recent clinical isolates of Enterococcus faecalis, Enterococcus faecium and Enterococcus avium.

Aminoglycoside modifying enzymes (AMEs) are major factors which confer aminoglycoside resistance on bacteria. Distribution of genes encoding seven AMEs was investigated by multiplex PCR for 279 recent clinical isolates of enterococci derived from a university hospital in Japan. The aac(6')-aph(2"), which is related to high level gentamicin resistance, was detected at higher frequency in Enterococcus faecalis (42.5%) than in Enterococcus faecium (4.3%). Almost half of E. faecalis and E. faecium isolates possessed ant(6)-Ia and aph(3')-IIIa. The profile of AME gene(s) detected most frequently in individual strains of E. faecalis was aac(6')aph(2") + ant(6)-Ia + aph(3')-IIIa, and isolates with this profile showed high level resistance to both gentamicin and streptomycin. In contrast, AME gene profiles of aac(6')-Ii+ ant(6)-Ia+aph(3')-IIIa, followed by aac(6')-Ii alone, were predominant in E. faecium. Only one AME gene profile of ant(6)-Ia+aph(3')-IIIa was found in Enterococcus avium. The ant(4')-Ia and ant(9)-Ia, which have been known to be distributed mostly among Staphylococcus aureus strains, were detected in a few enterococcal strains. An AME gene aph(2")-Ic was not detected in any isolates of the three enterococcal species. These findings indicated a variety of distribution profiles of AME genes among enterococci in our study site.

Analysis on distribution of insertion sequence IS431 in clinical isolates of staphylococci.

Distribution of insertion sequence IS431 in clinical isolates of Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus haemolyticus was investigated. Except for methicillin susceptible-S. aureus (MSSA), IS431 was detected in all isolates of the three staphylococcal species. In MSSA, only 20% of isolates with distinct coagulase types and genetic types possessed IS431. In a few MSSA isolates, an IS431 variant with internal deletion was found.

Genomic rearrangement of the mec regulator region mediated by insertion of IS431 in methicillin-resistant staphylococci.

Genomic diversification of the mec regulator region mediated by IS431 was investigated for clinical isolates of methicillin-resistant staphylococci. A single rearranged form of the mecR1 gene due to IS431 insertion was detected in the three staphylococcal species, while another type of mecR1 truncation with IS431 and an IS431 located downstream of mecI were found only in Staphylococcus haemolyticus. Genetic differentiation of IS431 and staphylococcal isolates suggested transmission of mecDNA with IS431-mediated rearrangement among different staphylococcal species.

Amplification of various genes of human group B rotavirus from stool specimens by RT-PCR.

BACKGROUND: The detection of the human group B rotavirus (HuGBR) CAL strain from India has given us an opportunity to design suitable primers for the detection of HuGBR since CAL is the second HuGBR detected until now, the Chinese Adult Diarrhoea Rotavirus (ADRV) being the first reported human pathogen belonging to this group of viruses. The primers described here may thus be used for the detection of human group B rotaviruses by reverse transcription-PCR (RT-PCR) in a diagnostic laboratory. OBJECTIVE: To establish a set of primers suitable for the detection of various genes of human group B rotaviruses using a rapid RT-PCR assay. STUDY DESIGN: Until recently, the Chinese ADRV strain was the only HuGBR strain that had been partially sequenced by cloning various viral genes using vector-specific primers. Consequently, there are very few reports in the literature describing primers that may be used for the detection of HuGBR viruses using RT-PCR in a clinical laboratory. The sequences of various genes from the ADRV strain that had been submitted to the nucleotide sequence database GenBank were analyzed in order to design several putative detection primer pairs for an RT-PCR assay. The rationale was to amplify the cognate genes from five isolates of the HuGBR CAL strain (CAL-1 to CAL-5) that have been detected to date from India. Primers that resulted in a specific product of the expected size from the CAL isolates were used to standardize a protocol for amplifying various genes of the CAL isolates under identical reaction conditions. RESULTS: Out of several synthetic oligonucleotides designed, 12 were found to be satisfactory for the amplification of gene segments 4, 5, 6, 7, and 9 from the five CAL isolates and are presented here. A set of previously described primers that have been shown to be specific for human group B rotavirus gene segment 8 were also found to amplify the cognate gene from the CAL isolates. All the reactions were carried out using the same thermal cycling conditions. CONCLUSIONS: The extreme virulence potential of HuGBR has been documented in several epidemics in China. Until recently, the Chinese ADRV strain was the only known HuGBr strain. As there have not been any reports of HuGBR infections outside China, there are no consensus nucleotide sequences available for HuGBR that may be used to validate primers for the detection of HuGBR. Here we report a set of 12 primer sequences that were designed from ADRV sequences and also found to amplify various genes from the different CAL isolates and hence may represent consensus primers suitable for the detection of HuGBR.

Rearrangement generated in double genes, NSP1 and NSP3, of viable progenies from a human rotavirus strain.

We generated rotavirus clones with rearrangement in vitro by serial passages of a human rotavirus strain (IGV-80-3) at high multiplicity of infection and determined nucleotide sequences of the rearranged genes from two distinct rotavirus clones, each of which possesses two rearranged genes: a common rearranged NSP1 gene and NSP3 gene with slightly different migration in polyacrylamide gel electrophoresis. Sequence analysis showed that the rearranged NSP1 and NSP3 genes had similar gene structures: concatemerization in a head to tail orientation and partial duplication of the open reading frame following the termination codon. The rearranged NSP1 gene had a direct repeat, whereas in the rearranged NSP3 gene, no such pattern was found.

New P serotype of group A human rotavirus closely related to that of a porcine rotavirus.

The VP7 and VP4 genes of two human group A rotavirus strains Mc323 and Mc345 with unique serologic and genomic properties, and isolated in Chiang Mai, Thailand, in 1989 [Urasawa et al. (1992) Journal of Infectious Diseases 166:227-234] were further characterized. The nucleotide and deduced amino acid sequences of the VP7 genes allowed the classification of both strains as serotype G9. The VP4 genes of both strains are 2,359 nucleotides in length and encode a protein of 775 amino acids like in most human rotaviruses. A comparison of the VP4 amino acid sequence of strain Mc323 with those of strain Mc345 and 24 human and animal rotaviruses representing 20 distinct VP4 genotypes reported to date showed that VP4 of Mc323 and Mc345 belong to genotype 19 previously reported for porcine rotavirus [Burke et al. (1994) Journal of General Virology 75:2205-2212]. To investigate the serological type (P serotype) of these VP4s, six reassortant viruses each containing a distinct VP4 gene characteristic of human rotaviruses and the VP7 gene of porcine rotavirus strain Gottfried (G4) were prepared, and antisera to these reassortants produced in rabbits. In neutralization tests, the P serotype of Mc323 was clearly differentiated from the five major P serotypes reported previously for human rotaviruses, suggesting that Mc323 and Mc345 represent a new human rotavirus P serotype tentatively called P11.

Distribution of insertion sequence-like element IS1272 and its position relative to methicillin resistance genes in clinically important Staphylococci.

The distribution of insertion sequence-like element IS1272 was analyzed for clinical isolates of Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus haemolyticus. In each of the staphylococcal species, IS1272 was detected in both methicillin-resistant (MR) and methicillin-susceptible strains of different genetic types. In MR isolates, IS1272 was generally located downstream of the truncated mecR1 gene (DeltamecR1), with an identical junction sequence occurring between DeltamecR1 and IS1272, although insertion of an additional gene sequence in the junction sequence was detected in one S. epidermidis isolate. These findings suggest that the mec element with the rearranged form of mecR1 (DeltamecR1-IS1272) has been transmitted to multiple clones of staphylococci.

Analysis on reassortment of rotavirus NSP1 genes lacking coding region for cysteine-rich zinc finger motif.

Rotavirus clones A5-10 and A5-16 isolated from a bovine rotavirus strain A5 possess NSP1 gene which has a point mutation generating a nonsense codon and a 500 base-deletion, respectively. As a result, the two A5 clones encode truncated NSP1 product which lacks cysteine-rich region forming zinc finger motif. In order to analyze reassortment of these mutated NSP1 gene with RNA segments from heterologous strains, we investigated a number of reassortant clones derived from coinfection with either A5-10, A5-16 or a reference strain A5-13 (possessing intact NSP1 gene) and either simian rotavirus SA11 or human rotavirus KU. In coinfection with SA11 and A5-13, selection rates of A5-13 segments in reassortants ranged approximately from 20 to 70% (46% for NSP1 gene). In contrast, in the reassortment between SA11 and A5-10 or between SA11 and A5-16, selection rates of NSP1 gene from A5-10 and A5-16 were only 1% (one clone) and 0%, respectively. In reassortants from crosses KU x A5-clones, selection rate of A5-13 NSP1 gene decreased to 15%, while 11 reassortants with A5-10 NSP1 gene (31%) and one reassortant with A5-16 NSP1 gene (2%) were isolated. Reassortants with A5-10 NSP1 possessed a single gene (segment 9 or 11) from KU in the genetic background of A5-10. One reassortant clone (cl-55) with A5-16 NSP1 gene possessed KU gene segments 3, 4, and 8-11. When single-step growth curves were compared, the reassortant cl-55 showed almost identical growth curve to that of KU, while KU showed a better replication than A5-16. These results indicated that although A5-10 or A5-16 NSP1 gene encoding the truncated NSP1 is selected into reassortants much less efficiently than normal NSP1 gene, the reassortants with the mutated NSP1 gene and RNA segments from heterologous strains normally replicated in cultured cells. Thus, cysteine-rich region of NSP1 was not considered essential for genome segment reassortment with heterologous virus.

Analysis of genomic diversity within the Xr-region of the protein A gene in clinical isolates of Staphylococcus aureus.

Protein A of Staphylococcus aureus contains a polymorphic Xr-region characterized by a tandem repeat of eight amino acid units. In this study, the diversity of genes encoding the repeat regions and their relatedness among S. aureus strains was analyzed. Ten different protein-A types characterized by repeat numbers 4-13 were identified in a total of 293 clinical isolates. The protein-A type with 10 repeat units (10 repeats) in the Xr-region was most frequently detected in methicillin-resistant S. aureus, whereas the majority of methicillin-susceptible strains were distributed almost evenly into protein-A types with 7-11 repeats. Strains that belonged to a single coagulase type were classified into multiple protein-A types, e.g. strains with the common coagulase types II and VII were differentiated into 7 and 8 protein-A types, respectively. Nucleotide sequence analysis of the Xr-region of 42 representative strains revealed the presence of 37 different genotypes (spa types), which were constituted by a combination of several of 24 different repeat unit genotypes. Based on the similarity in arrangement of repeat unit genotypes, 34 strains with different repeat numbers were classified into 5 genetic clusters (C1-C5). The clusters C1, C2 and C3 consisted exclusively of strains with identical coagulase types II, III, and IV, respectively. These findings suggested that the protein-A gene of S. aureus has evolved from a common ancestral clone in individual clusters independently.

Analysis of human rotavirus G serotype in Bangladesh by enzyme-linked immunosorbent assay and polymerase chain reaction.

Distribution of human rotavirus G serotype was investigated by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) with faecal specimens obtained from children with diarrhoea in Bangladesh. By ELISA, subgroup and G serotype were determined for 59.5% and 28.6% of group A rotavirus-positive specimens respectively. However, of the 120 specimens, the G serotype of which was not determined by ELISA, serotype of the 112 specimens was typed by PCR. In total, G serotype was assigned for 95.2% of all the specimens, showing the highest rate of G4 (41.7%), followed by G1 (23.2%) and G2 (14.9%). Twenty-four specimens showed mixed types, such as G2 with G1, G8 or G9, or G1 with G4. These results indicate that PCR combined with ELISA is highly effective for G serotyping of rotavirus.

Detection of group- and subgroup-specific antigens of bovine rotaviruses in Bangladesh.

The study was carried out to detect group- and subgroup-specific antigens of bovine rotaviruses. Stool specimens, collected from diarrhoeic calves of the Savar Dairy Farm, Bangladesh, were examined by an enzyme-linked immunosorbent assay, using group- and subgroup-specific monoclonal antibodies. Thirty-three specimens showed specificity for group A rotavirus. While subgrouping, 21 group A-positive specimens showed subgroup I specificity. Twelve specimens did not react with either of the subgroup I- and subgroup II-specific monoclonal antibodies.

Detection and characterization of novel rotavirus strains in the United States.

We recently established a rotavirus strain surveillance system in the United States to monitor the prevalent G serotypes before and after the anticipated implementation of a vaccination program against rotavirus and to identify the emergence of uncommon strains. In this study, we examined 348 rotavirus strains obtained in 1996 to 1997 from children with diarrhea in 10 U.S. cities. Strains were characterized for P and G types, subgroups, and electropherotypes by using a combination of monoclonal antibody immunoassay, reverse transcription-PCR, and hybridization. The four strains most commonly found worldwide comprised 83% of the isolates (P[8]G1, 66.4%; P[4]G2, 8.3%; P[8]G3, 6.9%; P[8]G4, 1.4%), but 9.2% were unusual strains (P[6]G9, 5.5%; P[8]G9, 1.7%; P[6]G1, 1.4%; and P[4]G1 and P[8]G2, 0. 3% each). Strains not typeable for P or G type accounted for 5.5% of the total, while 2.3% of the strains had more than one G type (mixed infections). All P[6]G9 strains tested had short electropherotypes and subgroup I specificity and were detected in 4 of 10 cities, while P[8]G9 strains had long electropherotypes and subgroup II VP6 antigens. Both sequence analysis of the VP7 open reading frame (about 94 to 95% amino acid identity with the VP7 gene of G9 prototype strain WI61) and binding to a G9-specific monoclonal antibody strongly suggest that U.S. G9 strains belong to serotype G9. The high detection rates of unusual rotaviruses with G9 (7.2%) or P[6] (6.9%) specificity in multiple U.S. cities suggest the emergence of new strains or inadequate diagnosis in the past. The epidemiologic importance of these strains remains to be determined.

Preferential selection of heterologous G3-VP7 gene in the genetic background of simian rotavirus SA11 detected by using a homotypic single-VP7 gene-substitution reassortant.

Introduction of segmented genomes into virion is an important process in viral replication of rotavirus. We previously studied the assortment of the VP7 gene segment (encoding outer capsid protein VP7) in the genetic background of simian rotavirus SA11 (G serotype 3, G3) and found the preferential selection of homologous G3 VP7 gene over VP7 gene of heterologous G serotype (G1, G2 or G4). In the present study, in order to clarify whether or not VP7 gene derived from different G3 rotavirus (heterologous G3-VP7 gene) is also preferentially selected in the SA11 background, a single-VP7 gene-substitution reassortant was prepared from SA11 through multiple steps of coinfection with rotaviruses in vitro. The isolated reassortant, SNR1, possessed VP7 gene derived from canine G3 rotavirus K9 and all other gene segments of SA11 origin, and showed an identical growth characteristic to that of SA11. Amino acid sequence of K9 VP7 gene showed a high degree of identity (93.6%) to SA11 VP7 gene. In analysis by mixed infection and multiple passages of SNR1 and a single VP7 gene (with G1, G2 or G4 specificity) reassortant in the SA11 background, the G3-VP7 gene became predominant at early passage numbers. However, in mixed infection with SA11 and SNR1, homologous G3-VP7 gene (SA11-VP7 gene) was preferentially selected into progenies over heterologous one (K9-VP7 gene). These results together with our previous findings suggested that G3-VP7 gene, irrespective of origin of species, was functionally adapted to the genetic background of SA11, although the homologous gene had a better fit with other SA11 genes than did heterologous one, providing suggestions for efficaciousness of multivalent reassortant rotavirus vaccine.

Serological and genomic characterization of human rotaviruses detected in China.

A total of 1,385 stool specimens were collected from children with diarrhea at two hospitals in Wuhan, Hubei Province, China, in 1994 and 1995, and screened for rotavirus by polyacrylamide gel electrophoresis of viral RNA. Group A rotavirus was detected with high frequency; 56.5% (87/154) and 40.8% (502/1,231) of the specimens collected in 1994 and 1995, respectively, were positive for rotavirus. Assignment of G serotype and P type (VP4 genotype) of group A rotavirus by ELISA with monoclonal antibodies and/or PCR, respectively, showed that strains of G2-P[4] and G1-P[8] specificity were predominant in 1994 and in 1995, respectively. In contrast, a single strain was found to have a P[9] type specificity, and no G4 strain was detected. Unusual combinations of RNA pattern-subgroup-G serotype-P type, such as long pattern-subgroup I-G1-P[8], short pattern-subgroup II-G3-P[4] and short pattern-subgroup I-G1-P[4], were detected in four specimens. Nucleotide sequences of the VP8* and/or NSP5 genes from two Chinese P[8] strains 470 and 582 and one Chinese P[9] strain 512 as well as five Japanese P[9] strains (K8, AU1, M318, 0264, and 0265) were determined and compared with the published sequences of the corresponding gene. In the phylogenetic tree of VP8* sequences of P[9] strains, which formed two clusters each having strain K8 or AU-1 as the representative strain, the Chinese P[9] strain was found in the cluster represented by AU-1, although it was most distantly related to other strains. While NSP5 sequences of human strains with P[9] specificity were related to simian and bovine strains, that of Chinese P[8] strains was most closely related to those of porcine strains. A single group C rotavirus (No. 208) was detected. Nucleotide sequences of its VP4, VP6, VP7, and NSP4 genes were very similar to those of group C human rotaviruses detected worldwide.

Analysis of diversity of mutations in the mecI gene and mecA promoter/operator region of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis.

Genomic diversity of mutation in the mecI gene or mecA promoter/operator region was analyzed for clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE). In most MRSA strains, a single base substitution was detected in either the mecI (three different positions) or the mecA promoter (two different positions), while a 28-base deletion in mecI was found in one strain. In contrast, no mutation was detected in these gene sequences of MRSE strains.

Survey of human group C rotaviruses in Japan during the winter of 1992 to 1993.

Fecal specimens from patients with acute diarrhea were collected from 10 prefectures in Japan over a 6-month period (November 1992 to April 1993), and the specimens that were negative for human group A rotaviruses were screened for the presence of human group C rotaviruses (CHRVs) by the reverse passive hemagglutination test. Of 784 specimens examined, 53 samples (6.8%) that were collected in 7 of 10 prefectures were positive for CHRV, indicating that CHRVs are widely distributed across Japan. Most of the CHRV isolates were detected in March and April, and CHRVs mainly prevailed in children ages 3 to 8 years. The genome electropherotypes of eight strains isolated in five individual prefectures were surprisingly similar to each other and were different from those of CHRV strains isolated to date. The outer capsid glycoprotein (VP7) gene homologies of the isolates retrieved in 1993 were subsequently analyzed by the dot blot hybridization method. As a result, the VP7 genes of the isolates revealed very high levels of homology not only with each other but also with the VP7 gene of the OK118 strain isolated in 1988. These results suggest that a large-scale outbreak of CHRV occurred during the winter of 1992 and 1993 in Japan.

Molecular discrimination of enterotoxin C subtype in clinical isolates of Staphylococcus aureus.

The subtype of staphylococcal enterotoxin C (SEC) of 33 S. aureus clinical isolates was determined by polymerase chain reaction and direct DNA sequencing of a portion of the SEC gene encoding the SEC subtype-specific region. With the exception of a single strain with the SEC2 gene, all other strains showing different biologic and genetic properties were proved to possess the SEC3 gene.